Hypoxia-mediated neurogenesis

ABSTRACT

Methods are described for the production of neurons or neuronal progenitor cells. Multipotent neural stem cells are proliferated in the presence of growth factors and erythropoietin which induces the generation of neuronal progenitor cells. The erythropoietin may be exogenously applied to the multipotent neural stem cells, or alternatively, the cells can be subjected to hypoxic insult which induces the cells to express erythropoietin.

FIELD OF THE INVENTION

[0001] This invention relates to methods of influencing multipotentneural stem cells to produce progeny that differentiate into neurons byexposing the stem cells and their progeny to erythropoietin.

BACKGROUND OF THE INVENTION

[0002] Neurogenesis in mammals is complete early in the postal period.Cells of the adult mammalian CNS have little or no ability to undergomitosis and generate new neurons. While a few mammalian species (e.g.rats) exhibit the limbed ability to generate new neurons in restrictedadult brain regions such as the dentate gyrus and olfactory bulb(Kaplan, J. Comp. Neurol., 195:323, 1981; Bayer, N.Y. Acad. Sci.,457:163, 1985), the generation of new CNS nerons in adult primates doesnot normally occur (Rakic, Science, 227:1054, 1985). This inability toproduce new nerve cells in most mammals (and especially primates) may beadvantageous for long-term memory retention; however, it is a distinctdisadvantage when the need to replace lost neuronal cells arise due toinjury or disease.

[0003] The role of stem cells in the adult is to replace cells that arelost by natural cell death, injury or disease. Until recently the lowturnover of cells in the mammalian CNS together with the inability ofthe adult mammalian CNS to generate new neuronal cells in response tothe loss of cells following injury or disease had led to the assumptionthat the adult mammalian CNS does not contain multipotent neural stemcells. The critical identifying feature of a stem cell is its ability toexhibit self-renewal or to generate more of itself. The simplestdefinition of a stem cell would be a cell with the capacity forself-maintenance. A more stringent (but still simplistic) definition ofa stem cell is provided by potten and Loeffler (Development, 110:1001,1990) who have defined stem cells as “undifferentiated cells capable ofa) proliferation, b) self-maintenance, c) the production of a largenumber of differentiated functional progeny, d) regenerating the tissueafter injury, and e) a flexibility in the use of these options.”

[0004] CNS disorders encompass numerous afflictions such asneurodegenerative diseases (e.g. Alzheimer's and Parkinson's), acutebrain injury (e.g. stroke, head injury, cerebral palsy) and a largenumber of CNS dysfunctions (e.g. depression, epilepsy, andschizophrenia). Degeneration in a brain region known as the basalganglia can lead to diseases with various cognitive and motor symptoms,depending on the exact location. The basal ganglia consists of manyseparate regions, including the striatum (which consists of the caudateand putamen), the globus pallidus, the substantia nigra, substantiainnominate, ventral pallidum, nucleus basalis of Meynert, ventraltegmental area and the subthalamic nucleus. Many motor deficits are aresult of neuronal degeneration in the basal ganglia. Huntington'sChorea is associated with the degeneration of neurons in the striatum,which leads to involuntary jerking movements in the host. Degenerationof a small region called the subthalamic nucleus is associated withviolent flinging movements of the extremities in a condition calledballismus, while degeneration in the putamen and globus pallidus isassociated with a condition of slow writhing movements or athetosis.Other forms of neurological impairment can occur as a result of neuraldegeneration, such as cerebral palsy, or as a result of CNS trauma, suchas stroke and epilepsy.

[0005] In recent years neurodegenerative disease has become an importantconcern due to the expanding elderly population which is at greatestrisk for these disorders. These diseases, which include Alzheimer'sDisease and Parkinson's Disease, have been linked to the degeneration ofneuronal cells in particular locations of the CNS, leading to theinability of these cells or the brain region to carry out their intendedfunction. In the case of Alzheimer's Disease, there is a profoundcellular degeneration of the forebrain and cerebral cortex. In addition,upon closer inspection, a localized degeneration in an area of the basalganglia, the nucleus basalis of Meynert, appears to be selectivelydegenerated. This nucleus normally sends cholinergic projections to thecerebral cortex which are thought to participate in cognitive functionsincluding memory. In the case of Parkinson's Disease, degeneration isseen in another area of the basal ganglia, the substantia nigra parcompacta. This area normally sends dopaminergic connections to thedorsal striatum which are important in regulating movement. Therapy forParkinson's Disease has centered upon restoring dopaminergic activity tothis circuit through the use of drugs.

[0006] In addition to neurodegenerative diseases, acute brain injuriesoften result in the loss of neurons, the inappropriate functioning ofthe affected brain region, and subsequent behavior abnormalities.

[0007] To date, treatment for CNS disorders has been primary via theadministration of pharmaceutical compounds. Unfortunately, this type oftreatment has been fraught with many complications including the limitedability to transport drugs across the blood-brain barrier and thedrug-tolerance which is acquired by patients to whom these drugs areadministered long-term. For instance, partial restoration ofdopaminergic activity in parkinson's patients has been achieved withlevodopa, which is a dopamine precursor able to cross the blood-brainbarrier. However, patients become tolerant to the effects of levodopa,and therefore, steadily increasing dosages are needed to maintain itseffects. In addition, there are a number of side effects associated withlevodopa such as increased and uncontrollable movement.

[0008] Recently, the concept of neurological tissue grafting has beenapplied to the treat of neurological diseases such as Parkinson'sDisease. Neural grafts may avert the need not only for constant drugadministration, but also for complicated drug delivery systems whicharise due to the blood-brain barrier. However, there are limitations tothis technique as well. First, cells used for transplantation whichcarry cell surface molecules of a differentiated cell from another hostcan induce an immune reaction in the host. In addition, the cells mustbe at a stage of development where they are able to form normal neuralconnections with neighboring cells. For these reasons, initial studieson neurotransplantation centered on the use of fetal cells. Severalstudies have shown improvements in patients with Parkinson's Diseaseafter receiving implants of fetal CNS tissue. Implants of embryonicmesencephalic tissue containing dopamine cells into the caudate andputamen of human patients was shown by Freed et al. (N Engl J Med327:1549-1555 (1992)) to offer long-term clinical benefit to somepatients with advanced Parkinson's Disease. Similar success was shown bySpencer et al. (N Engl J Med 3271541-1548 (1992)). Wiener et al. (N EnglJ Med 327:1556-1563 (1992)) have shown long-term functional improvementsin patients with MPTP-induced Parkinsonism that received bilateralimplantation of fetal mesencephalic tissue. Perlow, et al. describe thetransplantation of fetal dopaminergic neurons into adult rats withchemically induced nigrostriatal lesions in “Brain grafts reduce motorabnormalities produced by destruction of nigrostriatal dopamine system,”Science 204:643-647 (1979). These grafts showed good survival, axonaloutgrowth and significantly reduced the motor abnormalities in the hostanimals.

[0009] While the studies noted above are encouraging, the use of largequantities of aborted fetal tissue for the treatment of disease raisesethical considerations and political obstacles. There are otherconsiderations as well Fetal CNS tissue is composed of more than onecell type, and thus is not a well-defined source of tissue. In addition,there are serious doubts as to whether an adequate and constant supplyof fetal tissue would be available for transplantation. For example, inthe treatment of MPTP-induced Parkinsonism (Widner supra) tissue from 6to 8 fresh fetuses were required for implantation into the brain of asingle patient. There is also the added problem of the potential forcontamination during fetal tissue preparation. Moreover, the tissue mayalready be infected with a bacteria or virus, thus requiring expensivediagnostic testing for each fetus used. However, even diagnostic testingmight not uncover all infected tissue. For example, the diagnosis ofHIV-free tissue is not guaranteed because antibodies to the virus aregenerally not present until several weeks after infection

[0010] While currently available transplantation approaches represent asignificant improvement over other available treatments for neurologicaldisorders, they suffer from significant drawbacks. The inability in theprior art of the transplant to fully integrate into the host tissue, andthe lack of availability of neuronal cells in unlimited amounts from areliable source for grafting are, perhaps, the greatest limitations ofneurotransplantation. A well-defined, reproducible source of neuralcells has recently been made available. It has been discovered thatmultipotent neural stem cells, capable of producing progeny thatdifferentiate into neurons and glia, exist in adult mammalian neuraltissue. (Reynolds and Weiss, Science 255:1707 (1992)). Methods have beenprovided for the proliferation of these stem cells to provide largenumbers of neural cells that can differentiate into neurons and glia(See. U.S. Pat. No. 5,750,376, and International Application No. WO93/101275). Various factors can be added to neural cell cultures toinfluence the make-up of the differentiated progeny of multipotentneural stem cell progeny, as disclosed in published PCT application WO94/10292. Additional methods for directing the differentiation of thestem cell progeny would be desirable.

SUMMARY OF THE INVENTION

[0011] A method of producing neurons or neuronal progenitor cells whichcan be used for transplantation or other purposes is described. Themethod comprises inducing multipotent neural stem cells to produceneuronal progenitor cells by proliferating the multipotent neural stemcells in the presence of growth factors and erythropoietin. Theerythropoietin may originate from the population of neural cells bysubjecting the cells to hypoxic insult which induces neural cells toexpress erythropoietin. Alternatively, the erythropoietin may beprovided exogenously.

DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term multipotent or oligopotent neural stemcell refers to an undifferentiated cell which is capable ofself-maintenance. Thus, in essence, a stem cell is capable of dividingwithout limit. The non-stem cell progeny of a multipotent neural stemcell are termed “progenitor cells.” A distinguishing feature of aprogenitor cell is that, unlike a stem cell, it has limitedproliferative ability and thus does not exhibit self-maintenance. It iscommitted to a particular path of differentiation and will, underappropriate conditions, eventually differentiate. A neuronal progenitorcell is capable of a limited number of cell divisions before giving riseto differentiated neurons. A glial progenitor cell likewise is capableof a limited number of cell divisions before giving rise to astrocytesor oligodendrocytes. A neural stem cell is multipotent because itsprogeny include both neuronal and glial progenitor cells and thus iscapable of giving rise to neurons, astrocytes, and oligodendrocytes.

[0013] Various factors can be added to neural cell cultures to influencethe make-up of the differentiated progeny of multipotent neural stemcell progeny, as disclosed in WO 94/10292. It has now been found thaterythropoietin (EPO), a hormone thought to influence the differentiativepathway of hematopoietic stem cells and/or their progeny, can increasethe number of neuronal progeny that are generated from proliferatedmultipotent neural stem cells. Multipotent neural stem cellsproliferated in the presence of EPO produce a greater percentage ofneuronal progenitor cells than multipotent neural stem cellsproliferated in the absence of EPO.

[0014] Multipotent neural stem cells can be obtained from embryonic,juvenile, or adult mammalian neural tissue (e.g. mouse and otherrodents, and humans and other primates) and can be induced toproliferate in vitro or in vivo using the methods disclosed in publishedPCT application WO 93/01275 and U.S. Pat. No. 5,750,376. Briefly, theadministration of one or more growth factors can be used to induce theproliferation of multipotent neural stem cells. Preferredproliferation-inducing growth factors include epidermal growth factor(EGF), amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1),basic fibroblast growth factor (bFGF or FGF-2), transforming growthfactor alpha (TGFα), and combinations thereof. For the proliferation ofmultipotent neural stem cells in vitro, neural tissue is dissociated andthe primary cell cultures are cultured in a suitable culture medium,such as the serum-free defined medium described in Example 1. A suitableproliferation-inducing growth factor, such as EGF (20 ng/ml) is added tothe culture medium to induce multipotent neural stem cell proliferation.

[0015] In the absence of substrates that promote cell adhesion (e.g.ionically charged surfaces such as poly-L-lysine and poly-L-ornithinecoated and the like), multipotent neural stem cell proliferation can bedetected by the formation of clusters of undifferentiated neural cellstermed “neurospheres”, which after several days in culture, lift off thefloor of the culture dish and float in suspension. Each neurosphereresults from the proliferation of a single multipotent neural stem celland is comprised of daughter multipotent neural stem cells and neuralprogenitor cells. The neurospheres can be dissociated to form asuspension of undifferentiated neural cells and transferred to freshgrowth-factor containing medium. This reinitiates proliferation of thestem cells and the formation of new neurospheres. In this manner, anunlimited number of undifferentiated neural stem cell progeny can beproduced by the continuous culturing and passaging of the cells insuitable culture conditions.

[0016] Various procedures are disclosed in WO 94/10292 and U.S. Pat No.5,750,376 which can be used to induce the proliferated neural stem cellprogeny to differentiate into neurons, astrocytes and oligodendrocytes.To increase the number of neuronal progenitor cells that are produced bythe multipotent neural stem cells, the proliferating stem cells can beexposed to EPO. The EPO can be exogenously added at concentrations fromabout 0.1 to 10 units/ml. Alternatively, the neural cells can be inducedto express endogenous EPO by subjecting the cells to hypoxic insult.Subsequent differentiation of the progenitor cell progeny results in atleast a two-fold increase in the numbers of neurons generated comparedto progeny of stem cells that have not been exposed to EPO, as evidencedby immunocytochemical analysis. Differentiation of cells that have notbeen exposed to endogenously added EPO or hypoxic insult typicallyresults in a population of cells containing about 3% neurons. Thepercentage of neurons increases to about 6% with hypoxia treatment, andto about 10% with exposure to exogenous EPO, with the percentage ofastrocytes and oligodendrocytes remaining about the same as the controlpeons.

[0017] Washout experiments, in which the growth factor/EPO medium isremoved after 24 hours and changed to regular growth factor-containingmedium, reveals that the EPO instructs the stem cells prior to theirfirst cell division, to produce more neurons. The continued presence ofEPO after the initial 24 hours does not result in a further increase inthe numbers of neurons over cultures subjected to EPO for a 24 hourperiod.

[0018] The ability to manipulate the fate of the differentiative pathwayof the multipotent neural stem cell progeny to produce more neuronalprogenitor cells and neurons is beneficial. Cell cultures that contain ahigher percentage of neuronal progenitor cells and/or neurons will beuseful for screening the effects of various drugs and other agents onneuronal cells. Methods for screening the effect of drugs on cellcultures are well known in the art and are also disclosed in U.S. Pat.No. 5,750,376.

[0019] Cell cultures with an enriched neuronal-progenitor cell and/orneuron population can be used for transplantation to treat variousneurological injuries, diseases or disorders. The neuronal progenitorcells or neurons or a combination thereof can be harvested andtransplanted into a patient needing neuronal augmentation. Neuronalprogenitor cells are particularly suitable for transplantation becausethey are still undifferentiated and, unlike differentiated neurons,there are no branched processes which can be damaged duringtransplantation procedures. Once transplanted, the neuronal progenitorcells differentiate in situ into new, functioning neurons. Suitabletansplantation methods are known in the art and are disclosed in U.S.Pat. No. 5,750,376.

[0020] Alternatively, a patient's endogenous multipotent neural stemcells could be induced to proliferate in situ to produce neuronalprogenitor cells by administering to the patient a condition comprisingone or more growth factors which induces the patient's neural stem cellsto proliferate and EPO which instructs the proliferating neural stemcells to produce neo progenitor cells which eventually differentiateinto neurons. Suitable methods for administering a composition to apatient which induces the in situ proliferation of the patient's stemcells are disclosed in U.S. Pat. No. 5,750,376.

[0021] All cited references, patents and applications are hereinincorporated in their entireties by reference.

EXAMPLE 1 Multipotent Neural Stem Cell Proliferation

[0022] Striata from 14 day-old mouse embryos were removed using sterileprocedure. Tissue was mechanically dissociated into serum-free mediumcomposed of a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM)and F-12 nutrient (Gibco). Dissociated cells were centrifuged, thesupernatant aspirated, and the cells resuspended at a concentration ofabout 1×10⁵ cell/ml in a serum-free medium, referred to herein as“complete medium” composed of DMEM/F-12 (1:1) including glucose (0.6%),glutamine (2 μM), sodium bicarbonate (3 mM), and HEPES(4-[2hydroxyethyl]-1-piperazineethanesulfonic acid) buffer (5 mM) (allfrom Sigma except glutamine [Gibco]). A defined hormone mix and saltmixture (Sigma) that included insulin (25 μ/ml), tansferrin (100 μ/ml),progesterone (20 nM), putrescine (60 μM), and selenium chloride (30 nM)was used in place of serum. The complete medium was supplemented with 20ng/ml of EGF (Collaborative Research). Cells were seeded in a T25culture flask and housed in an incubator at 37° C., 100% humidity, 95%air/5% CO₂. Stem cells within the cultures began to proliferate within3-4 days and due to a lack of substrate lifted off the floor of theflask and continued to proliferate in suspension forming neurospheres.

EXAMPLE 2 Hypoxia-Induced Neurogenesis

[0023] After 6 days in vitro primary neurospheres formed using themethods described in Example 1 were dissociated and were replated inEGF-containing medium. After 24 hours, the cells were exposed to amodest hypoxic insult by decreasing the concentration of oxygen in theculture medium for varying lengths of time (from 1 to 12 hours) fromnormal levels of 135 mmHg to 30-40 mmHg. The cells were then cultured inthe EGF-containing complete medium described in Example 1 in 95% air/5%CO₂ for 7 days. Hypoxia did not prevent multipotent neural stem cellproliferation, as evidenced by the formation of secondary neurospheres.The number of progeny produced from hypoxia-treated stem cells was thesame as that in control cultures not subjected to hypoxic insult.

[0024] Secondary neurospheres generated from untreated orhypoxia-treated stem cells were dissociated into single cells andinduced to differentiate by plating between 0.5×10⁶ and 1.0×10⁶ cellsonto poly-L-ornithine-coated (15 μ/ml) glass coverslips in 24 wellNuclon (1.0 ml/well) culture dishes in EGF-free complete mediumoptionally supplemented with 1% FBS. After 7 days, the cells wereassayed using immunocytochemical analysis for the presence of neurons.Cultures that had been subjected to hypoxic conditions for 1 to 4 hourshad approximately a two-fold increase in the percentage of neurons(approx. 6%) over control cultures (approx. 3%). Cultures subjected to 4to 8 hours of hypoxia had fewer neurons produced and cultures subjectedto about 12 hours of hypoxia had normal levels (approx. 3%). The hypoxicinsult induced a rapid up-regulation of hypoxia-induced factor (HIF) inthe multipotent neural stem cell progeny. HIP is a transcription factorfor EPO . The 4 hour hypoxia-induced increase in neurogenesis could beblocked by the addition of an EPO-neutralizing antibody at 3 μ/ml.

EXAMPLE 3 Erythropoietin-Induced Neurogenesis

[0025] After 6 days in vitro primary neurospheres formed using themethods described in Example 1 were dissociated and replated in completemedium containing EGF at 20 ng/ml and human recombinant EPO at 0.1 to 10units/ml for either 24 hours or 7 days under normal oxygen conditions(95% air/5% CO₂; 135 mmHg). In both cases, immunocytochemistry revealedan EPO dose-dependent three-fold increase in the numbers of neuronsgenerated.

We claim:
 1. A method of inducing the differentiation of a multipotentneural stem cell culture into neurons, said method comprising the stepsof: (a) inducing said multipotent neural stem cell culture toproliferate; (b) exposing said culture to hypoxic conditions; and (c)allowing said culture to differentiate into a differentiated cellpopulation containing neurons, wherein said differentiated cellpopulation is enriched in neurons compared to when step (b) above is notperformed.